Poster Z1 - Developing Multistage Fluidic Chips for Muscle and Neuron Co-Culture

Introduction: In vivo studies by our lab and others have shown that muscle exercise is correlated with improved motor function and control in both physiological and pathological states, indicating a potential role for muscle fiber contraction in modulating motor neuron growth [1]. However, as cell-specific signaling can be hard to isolate and control in vivo, the relationship between motor neuron recruitment and muscle activation still is understudied due to lack of relevant mesoscale in vitro model systems. We have designed and fabricated a multistage modular neuromuscular fluidic chip design consisting of a central “spinal cord”, containing independently controllable motor neuron spheroids, and a distal “limb”, containing mature muscle tissue. State-of-the-art neuromuscular co-culture microfluidic systems only enable monitoring single motor units and isolate motor neurons and skeletal muscle into fluidically separate compartments, thereby minimizing biochemical and mechanical communication. By contrast, our meso-fluidic open-well chip enables monitoring multiple independent motor units in parallel and encourages biochemical and mechanical crosstalk between skeletal muscle and motor neurons, as we have shown both to be modulated by exercise [2]. In the current study, we describe the design and fabrication of such meso-scale co-culture chips, as well as preliminary studies on the viability of housing cells within the chips.

Materials and

Methods: The stages of the co-culture chip are fabricated using Surgical Resin V1 on a Form 3B SLA printer (Formlabs). 24 hours post cure, the co-culture chips are sterilized with a Tuttnauer benchtop line autoclave at 121˚C using the preset plastic cycle. The main bodies of the chips are adhered to glass slides in a 6 well plate using KwikSil™ silicon adhesive (World Precision Instruments). Fibrin gel, consisting of fibrinogen from bovine plasma (Sigma Aldrich, F8630) and thrombin from bovine plasma (Sigma Aldrich, T4648) dissolved in muscle growth media with IGF and ACA, is molded into a micro-grooved 1 mm layer at the bottom of the chip as shown in Figure 1a using a surgical resin stamp stage with a printed UpPhoto Resin (UpNano) stamp adhered to the bottom (Figure 1b). The stamp stage is coated with 1% Bovine Serum Albuminate (Thermo Fisher Scientific, 37525) to help with separating the stage from fibrin. Post fibrin curing, the stamp stage is removed and then seeded with a solution of C2C12 myoblast cells [3] separated and blocked from the neuron channels with a 10:1 PDMS (polydimethylsiloxane, Sylgard Dow) gate. The growth, differentiation, and stimulated mechanical response of the C2C12 cells is then observed to determine the viability of culturing live cells with the platform. Subsequently, HBG3 neuron spheroids, prepared as described in [2], are placed in the separated neuron wells depicted in Figure 1c and tested for their growth rate while sharing the same chip and media as the C2C12 tissue cultures.

Results, Conclusions, and Discussions: The design of the co-culture chip from this work facilitates physical but not fluidic separation of tissue engineered murine muscle and motor neurons, thus preserving biochemical and mechanical crosstalk between the different cell types. The chips show promise for promoting studies on the correlation between muscle exercise and neuron growth, enabling us to study how muscle-secreted cytokines and dynamic movement during exercise impact motor neuron growth and maturation. The “Spinal Cord” mesofluidic chips described in this study could be used to quantify the relationship between motor neuron growth and muscle exercise, lending mechanistic insight to our prior in vivo observations. Furthermore, the design of the chips described, if mirrored, lend themselves to a scalable spinal cord platform with two or more muscle wells or “limbs” that could be used to comparatively study neurite extension towards different muscle tissue pathological and physiological states (see Figure 1f and Figure 1g). We will continue to assess the viability of such chip platforms for co-culturing 2D muscle tissue and neuron cultures holding multiple independent motor units. Future studies will provide insight into the role of muscle exercise and injury in supporting neurite growth and will validate the platform for supporting a wider range of neuron and muscle co-culture studies quantitatively.

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